Background: It has been reported that heme oxygenase-1 (HO-1) is critical for tolerogenic dendritic cells (tolDCs) to suppress T cell responses and tolDCs will lost their immunoregulatory effects when HO-1 is blocked. Therefore, significant upregulation of HO-1 may markedly improve the tolerogenic capacity of tolDCs.

Methods: Bone marrow-derived DCs (BMDCs) were generated from Balb/c mice with low doses of GM-CSF and IL-4. The adherent immature BMDCs were obtained as TolDCs. Upregulation of HO-1 in TolDCs (HO-1hi-TolDCs) was achieved by CoPP treatment. SnPP-treated TolDCs served as control cells. LPS was used to induce DC maturation. T cell proliferation was stimulated by anti-CD3/CD28 antibodies. Adoptive transfer of Balb/c donor-derived DCs (5×106) to C57BL/6 recipient mice was performed 7 days prior to cardiac transplantation. To study the distribution and location of adoptively transferred donor DCs in recipient mice, these cells were labeled with cellvue claret in advance.

Results: CoPP treatment dramatically increased HO-1 expression in TolDCs, which rendered TolDCs refractory to LPS-induced maturation, enhanced their capability to suppress both CD4+ and CD8+ T cell proliferation stimulated by anti-CD3/CD28 antibodies (Fig.1), as well as induce more allogenic regulatory T cells (Tregs) in vitro after the coculture with naïve CD4+ T cells from C57BL/6 mice. Adoptive transfer of donor-derived untreated TolDCs significantly prolonged cardiac allograft survival compared to untreated control group (18.000±2.853 vs. 7.500±0.289 days). Interestingly, adoptive transfer of CoPP-treated TolDCs further extended the prolongation of allograft survival (36.778±6.974 days) (P<0.01, vs. untreated TolDCs group). In contrast, adoptive transfer of SnPP-treated TolDCs almost had no effects on the allograft survival (9.143±0.670 days) (Fig.2). Furthermore, adoptively transferred TolDCs were found to be mainly distributed in the recipient spleens. CoPP-treated donor TolDCs were found to be able to keep high levels of HO-1 expression and can survive longer than untreated TolDCs after adoptive transfer. In addition, alloreactive T cell response was significantly inhibited in CoPP-treated TolDCs group at day 7 after transplantation.

Conclusion: In vitro generated HO-1hi-TolDCs have enhanced tolerogenic capacity to modulate alloimmune responses both in vitro and in vivo, thus may provide an antigen-specific and cost-effective novel strategy to induce transplant tolerance.